In the last few years, there became known from the technical and patent literature numerous processes for attachment of enzymes to water-insoluble carriers. A summarizing survey has been made, for example, by R. A. Messing (editor): Immobilized Enzymes for Industrial Reactors, Academic Press, New York, 1975. According to the present state of knowledge, there may be considered 4 ways of attaching an enzyme for making it insoluble: (1) adsorption, (2) ionic binding, (3) encapsulation, and (4) covalent binding to a carrier or cross-linking of the enzymes. Combinations of these basic types of immobilization are also known.
Enzymes linked by adsorption to an enzyme-free carrier, such as activated charcoal or polysaccharides, have the disadvantage that, because of the relatively weak adsorptive attachment, desorption readily occurs. Especially, if changes in the concentration of ions and of temperature occur, a detachment of the adsorption bond may easily take place, and thus the so-called "bleeding-out" of the enzymes.
In case of an ionic binding of the enzyme to a polyanionic or polycationic carrier (as for example ion exchange resins), there exists as well the disadvantage of a relatively weak binding between the polyionic carrier and the enzyme, because the enzyme contains only weakly ionic groups, as a rule. The ion exchange effect of the enzymes thus attached, which leads to the unintended removal of certain ions from a beverage in the course of a treatment of beverages, for example, is a decisive disadvantage which interferes with many reactions.
Enzymes which are encapsulated into polymeric substances (e.g. cross-linked polyacrylamide), have as their main disadvantage the relatively difficult diffusion through the molecular sieve of the inclusion material. The apparent Michaelis constant of an enzyme thus encapsulated is increased thereby, compared even to lower-molecular substrates. In addition, there is the danger that the encapsulated enzymes, due to their elasticity penetrate through the pores of the inclusion material and thus "bleed out."
In the fourth type of attachment mentioned above, the enzyme is covalently and therefore very firmly linked to a reactive group of a water-insoluble carrier. By far the majority of the publications relating to the fixation of enzymes, and the present patent application as well, are concerned with this type of immobilization. A whole range of known immobilized enzyme compositions thus produced are not useful from an economic-technical point of view, however, because they require the use of unduly expensive coupling reagents or carrier substances or excessively costly production processes. In the coupling processes presently know, for instance, a considerable excess of enzymes must be employed, since the major portion of the enzymes is inactivated during the coupling procedure. Most of the processes allow only for a small ratio of enzymes per carrier substance, because the carrier substances can only be covered with enzyme protein on their surface. Partial remedy can be obtained by pulverization (micronization) of the finished products, but these preparations, due to their fineness, can only be passed with difficulties by a stream of liquid, when used in a packed-bed reactor. A further disadvantage of most of the processes for immobilization of enzymes is that the carrier substances, such as glass or diatomaceous earth, are restricted to certain shapes and that it is impossible to give them a ball, splinter or membrane shape or use them as a cover for other materials, such as screens. Some of the carrier substances and coupling reagents are also of doubtful value because of their toxicity.
Several of the above-mentioned disadvantages of known fixed enzymes have been overcome for non-proteolytic enzymes, according to prior art, by coupling these enzymes, for example with glutardialdehyde, to collagen. The preparations thus produced, however, are easily attacked by microbes, and their specific activity is low. A similar situation exists with preparations obtained by cross-linking enzymes with a gel-forming protein (cf. German Offenlegungsschrift No. 2,246,002).
Here, a homogeneous mixture is formed, because enzymes and the gel-forming protein are crosslinked by random. These preparations are more stable towards microbes, but their efficacy is poor, because the enzymes are homogeneously distributed over the whole cross-section of the particle. With respect to high activity, cross-linking can only occur relatively weakly. This leads to such soft preparations that, when used in a packed bed, blocking and bleeding out of the enzymes take place. On the other hand, in case of a stronger cross-linking, this leads to inactivation due to excessively strong bonding and due to the inclusion of internal enzyme molecules. A considerable disadvantage of the last-mentioned fixing processes is also the fact that they cannot be used for proteolytic enzymes.